A novel universal method for measuring enzymatic activity using micro-calorimetry is applied to the HIV-1 protease (PR). Biocalorimetric assays measure the rate of heat evolution during conversion of substrate to product. Calorimetry thus gives a direct measure of the rate of an enzymatic reaction, as opposed to conventional assays where rate is calculated as a derivative of a quantity with time. Since no chromophore is necessary, the physiological substrates can be used. The activity of PR mutant (Q7K, L33I, L63I) was assayed using: 1) a standard chromogenic substrate (Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2); 2) a non-chromogenic version of the same peptide (Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2); and, 3) a non-chromogenic physiological substrate (Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2). The activity of PR as a function of salt concentration, pH, temperature (and <Cp>) for Km and Vmax are described. In addition, the inhibition by products pepstatin and acetyl-pepstain is described.